WO2004072230A2

WO2004072230A2 - Real-time polymerase chain reaction using large target amplicons

Info

Publication number: WO2004072230A2
Application number: PCT/US2004/002012
Authority: WO
Inventors: Keith Mckenney; Lidja Gillmeister; Kristina Marlowe; David Armistead
Original assignee: Clearant, Inc.
Priority date: 2003-02-10
Filing date: 2004-02-10
Publication date: 2004-08-26
Also published as: US20040170981A1; WO2004072230A3

Abstract

The present invention relates to methods for analyzing a target nucleic acid sequence in a biological material. More particularly, the present invention relates to methods for analyzing a target nucleic acid sequence by real time polymerase chain reaction using nucleic acid primers that are separated by at least about 750 nucleic acid residues in the target sequence.

Description

REAL-TIME POLYMERASE CHAIN REACTION USING LARGE TARGET AMPLICONS

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates to metiiods for analyzing a target nucleic acid sequence

in a biological material. More particularly, the present invention relates to methods for

analyzing a target nucleic acid sequence by real time polymerase chain reaction using nucleic

acid primers that are separated by at least about 750 nucleic acid residues in the target sequence.

2. Background of the Related Art

PCR (polymerase chain reaction) is a method for increasing the concentration of a

segment of a target sequence in a mixture of nucleic acid sequences wid out cloning or

purification. (See K. B. Mullis et al., U.S. Pat. Nos. 4,683,195 and 4,683,202).

This process for amplifying the target sequence consists of introducing two

oligonucleotide primers to the sample containing the desired target nucleic acid sequence,

followed by thermal cycling in the presence of a DNA polymerase. The two primers are

complementary to their respective strands of the target sequence. To effect amplification,

the genetic material within the sample is first denatured and then the primers are annealed to

their complementary sequences within d e target molecule. Following annealing, d e primers

are extended with a polymerase so as to form a new pair of complementary strands.

The steps of denaturation, annealing and extension can be repeated many times (i.e.,

denaturation, annealing and extension constitute one "cycle"; there can be numerous "cycles") to obtain a high concentration of an amplified segment of the desired target

sequence. The length of the amplified segment of the desired target sequence is determined

by the relative p ositions of the primers with respect to each other, and therefore, this length is a controllable parameter. Because the desired amplified segments of the target sequence

become d e predominant sequences (in terms of concentration) in the mixture, they are said

to be "PCR amplified".

With PCR, it is possible to amplify a single copy of a specific target sequence in

genomic DNA to a level detectable by several different methodologies (e.g., hybridization

with a labelled probe; incorporation of biotinylated primers followed by avidin-enzyme

conjugate detection; incorporation of ³²P-labelled deoxynucleotide triphosp hates, e.g., dCTP

or dATP, into die amplified segment). In addition to genomic DNA, any oligonucleotide

sequence can be amplified with the appropriate set of primer molecules.

End-point PCR is a polynucleotide amplification protocol. The amplification factor

that is observed is related to the number (n) of cycles that have occurred and the efficiency

of replication at each cycle (E), which, in turn, is a function of the priming and extension

efficiencies during each cycle. Amplification has been observed to follow the form Eⁿ, until

high concentrations of the PCR product have been made.

At d ese high product concentrations, d e efficiency of replication tends to drop

significandy. It has been suggested that this is probably due to the displacement of the

primers by the longer complementary strands o f the PCR product. At concentrations in

excess of 10"⁸ M, the rate of the two complementary PCR amplified product strands finding

each other during the prkαing reactions becomes sufficiently fast that it may occur before or concomitantiy with the extension step of the PCR process. This ultimately leads to a

reduced priming efficiency, and, consequendy, a reduced cycle efficiency. Continued cycles

of PCR lead to declining increases of PCR product molecules, until the PCR product

eventually reaches a plateau concentration (the "end-point"), usually a concentration of

approximately 10"⁸ M. As a typical reaction volume is about 100 microliters, this

corresponds to a yield of about όxlO¹¹ double stranded product molecules.

Real-time PCR is also a polynucleotide amplification protocol, but PCR product

analysis occurs simultaneously with amplification of the target sequence. Detecting agents,

such as DNA dyes or fluorescent probes, can be added to the PCR mixture before

amplification and used to analyze PCR products during amplification. Sample analysis

occurs concurrently with amplification in the same tube within the same instrument. This

combined approach decreases sample handling, saves time, and greatiy reduces the risk of

product contamination, as there is no need to remove the samples from their closed

containers for further analysis. The concept of combining amplification with product

analysis has become known as "real-time" or "quantitative" PCR. (See, e.g., WO/9746707 A2,

WO/9746712A2 and WO/9746714A1).

Originally, monitoring fluorescence each cycle of PCR involved die use of ethidium

bromide. See Higuchi et al., "Simultaneous amplification and detection of specific DNA

sequences," Bio /Technology 10:413-417 (1992); Higuchi et al, "Kinetic PCR analysis: real time

monitoring of DNA amplification reactions," Bio /Technology 11:1026-1030 (1993). In dαat

system, fluorescence was measured once per cycle as a relative measure of product

concentration. Etiiidium bromide detects double stranded DNA; thus, if the desired target

nucleic acid sequence is present, fluorescence intensity increases with temperature cycling (otherwise no fluorescence). Furthermore, the cycle number where an increase in

fluorescence is first detected increases inversely proportionally to the log of the initial target

sequence concentration. Other fluorescent systems have since been developed that are capable of providing additional data concerning the nucleic acid concentration.

A significant limitation in the use of real-time PCR is the lengdi of the target nucleic

acid sequence. That is, as the target amplicon length increase, the efficiency of real-time

PCR decreases. Practical limits for target amplicon lengdi in most commercially available

PCR systems are generally less ti an 500 bp, usually in the range of 80-200 bp. Larger

amplicons have been obtained by some, but to date there remains a need for routinely

amplifying large target sequences in real time PCR.

Each of the above refe ences is incorporated by reference herein where appropriate

for teachings of additional or alternative details, features and/ or technical background.

SUMMARY OF THE INVENTION

An object of the invention is to solve at least the problems and/or disadvantages of

the relevant art, and to provide at least the advantages described hereinafter.

Accordingly, it is an object of the present invention to provide metiiods for analyzing

a target nucleic acid sequence by real time polymerase chain reaction using nucleic acid

primers that are separated by at least about 750 nucleic acid residues in the target sequence.

Other objects, features and advantages of the present invention will be set forth in the

detailed description of preferred embodiments that follows, and in part will be apparent

from die description or may be learned by practice of the invention. These objects and advantages of the invention will be realized and attained by the compositions and metiiods

particularly pointed out in the written description and claims hereof.

In accordance with these and other objects, a first embodiment of the present invention is directed to a method for analyzing a target nucleic acid sequence, comprising: (i)

adding to a biological material an effective amount of at least two nucleic acid primers that

hybridize under stringent conditions to predetermined sequences of the target sequence and

are separated by at least about 750 nucleic acid residues, (ii) amplifying the target nucleic acid

sequence by a polymerase chain reaction which comprises adding a polymerase to the

biological material and primers to form an amplification mixture and thermally cycling the

amplification mixture between at least one denaturation temperature and at least one

elongation temperature, and (iii) detecting and quantifying said target nucleic acid sequence.

According to this embodiment of the present invention, during the thermal cycling, the

elongation temperature is not more than about 70°C and the denaturation temperature is not

more tiian about 95°C, and the amplification mixture is maintained at the denaturation

temperature for a period of not more than about 30 seconds and at the elongation

temperature for a period of not less than about 1 minute.

Additional advantages, objects, and features of the invention will be set ford in part

in the description which follows and in part will become apparent to those having ordinary

skill in the art upon examination of the following or may be learned from practice of the

invention. The objects and advantages of the invention may be realized and attained as

particularly pointed out in the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be described in detail with reference to the following drawings in

which like reference numerals refer to like elements wherein:

Figure 1 shows forward and reverse primers useful in preparing large target amplicons based on die genomic nucleic acid sequence of human Parvovirus B19 (SEQ ID

NO.: 1).

Figure 2 shows forward and reverse primers useful in preparing large target

amplicons based on the genomic nucleic acid sequence of hepatitis B virus (SEQ ID NO.:

2).

Figure 3 shows forward and reverse primers useful in preparing large target

amplicons based on die genomic nucleic acid sequence of porcine parvovirus (SEQ ID NO.:

3)-

Figure 4 shows forward and reverse primers useful in preparing large target

amplicons based on the genomic nucleic acid sequence of Sindbis virus (SEQ ID NO.: 4).

Figure 5 shows forward and reverse primers useful in preparing large target

amplicons based on the genomic nucleic acid sequence of West Nile virus (SEQ ID NO.: 5).

Figures 6A and 6B show forward and reverse primers useful in preparing large target

amplicons based on the genomic nucleic acid sequence of the 16S ribosomal RNA gene

(SEQ ID NO.: 6) and the 23S ribosomal RNA gene of Es heήchia coli (SEQ ID NO.: 7).

Figures 7A and 7B show forward and reverse primers useful in preparing large target

amplicons based on the genomic nucleic acid sequence of the 18S ribosomal RNA gene

(SEQ ID NO.: 8) and the 25S ribosomal RNA gene of yeast (S. cerevisiae) (SEQ ID NO.: 9). Figure 8 shows forward and reverse primers useful in preparing large target amplicons based on the nucleic acid sequence of human mitochondrial DNA (SEQ ID NO.:

10).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

A. Definitions

Unless defined otiierwise, all technical and scientific terms used herein are intended

to have die same meaning as is commonly understood by one of ordinary skill in the relevant

art.

As used herein, the singular forms "a," "an," and "the" include the plural reference

unless the context clearly dictates otherwise.

As used herein, the term "biological material" is intended to mean any substance

derived or obtained from a living organism. Illustrative examples of biological materials

include, but are not limited to, the following: cells; tissues; blood or blood components;

proteins, including recombinant and transgenic proteins, and proetinaceous materials;

enzymes, including digestive enzymes, such as trypsin, chymotrypsin, alpha-galactosidase and

iduronodate-2-sulfatase; immunoglobulins, including mono and polyimmunoglobulins;

botanicals; food and the like. Preferred examples of biological materials include, but are not

limited to, the following: ligaments; tendons; nerves; bone, including demineralized bone

matrix,' grafts, joints, femurs, femoral heads, etc.; teeth; skin grafts; bone marrow, including

bone marrow cell suspensions, whole or processed; heart valves; cartilage; corneas; arteries

and veins; organs, including organs for transplantation, such as hearts, livers, lungs, kidneys,

intestines, pancreas, limbs and digits; lipids; carbohydrates; collagen, including native, afibrillar, atelomeric, soluble and insoluble, recombinant and transgenic, both native

sequence and modified; chitin and its derivatives, including NO-carboxy chitosan ( OCC); stem cells, islet of Langerhans cells and other cells for transplantation, including genetically altered cells; red blood cells; white blood cells, including monocytes; and platelets.

Additional examples of biological materials include forensic samples, human or animal

remains, stomach contents, mummified remains of a once-living organism, fossilized

remains, a product of manufacture containing or previously in contact with a biological

material, and fomites.

B. Particularly Preferred Embodiments

A first particular preferred embodiment of the present invention is directed to a method for analyzing a target nucleic acid sequence in a biological material, comprising:

(i) adding to a biological material an effective amount of at least two nucleic acid

primers, wherein these nucleic acid primers hybridize under stringent conditions to two

predetermined nucleic acid sequences of the target nucleic acid sequence that are separated

by at least about 750 nucleic acid residues,

(ii) amplifying the target nucleic acid sequence by a polymerase chain reaction

which comprises adding a polymerase to the biological material and primers to form an

ampKfication mixture and then thermally cycling the amplification mixture between at least

one denaturation temperature and at least one elongation temperature; and

(ϋi) detecting and quantifying said target nucleic acid sequence. According to this preferred embodiment of the present invention, die elongation temperature is not more than about 70°C and the denaturation temperature is not more tiian about 95°C. Additionally, according to tiiis preferred embodiment of d e present invention, during each thermal cycle, the amplification mixture is maintained at the denaturation

temperature for a period of not less than about 1 minute.

According to preferred embodiments of the present invention, the target nucleic acid

sequence preferably contains between about 500 and about 50,000 nucleic acid residues.

More preferably, die target nucleic acid sequence contains between about 1000 and about

10,000 nucleic acid residues, even more preferably between about 2000 and about 5000

nucleic acid residues and most preferably between about 2500 and about 5000 nucleic acid

residues.

The nucleic acid primers are each selected based on their ability to generate the

desired target nucleic acid sequence under the appropriate PCR conditions. Accordingly,

each primer must be specific for the desired target nucleic acid sequence. Similarly, each

primer must be selected so that they are not self-complementary or complementary to

another primer (or probe, if present).

According to preferred embodiments of the present invention, the sequences on the

target sequence d at correspond to the two primer sequences are separated by at least 750

nucleic acid residues. Preferably, the sequences which correspond to die primers are

separated by at least about 1000 nucleic acid residues, more preferably at least about 2000

nucleic acid residues, even more preferably at least about 3000 nucleic acid residues, still even more preferably at least about 4000 nucleic acid residues and most preferably at least about 5000 nucleic acid residues. According to an alternative embodiment of the present invention, the sequences on the target sequence that correspond to the two primer

sequences are separated by only about 500 nucleic acid residues.

The polymerase chain reaction employed in the inventive metiiods is performed

according to the methods and techniques known to those skilled in the art, i.e., a nucleic acid

primer pair is added to the biological material containing the sequence of interest to form an

amplification mixture that is then tiiermally cycled for a sufficient period of time to amplify

die desired sequence. The thermal cycling generally comprises cycling the amplification

mixture between at least one denaturation temperature and at least one elongation

temperature. Preferably, the ti ermal cycling comprises cycling d e amplification mixture

between at least one denaturation temperature, at least one annealing temperature and at

least one elongation temperature.

Specific temperatures for use in denaturation, elongation and/ or annealing may be

determined empirically by one skilled in the art based, for example, on the specific target

sequence being amplified and the particular probes employed. Likewise, the specific time(s)

that the amplification mixture is maintained at the various denaturation, elongation and/or

annealing temperature(s) may be determined empirically by one skilled in the art based on

similar considerations.

According to particularly preferred embodiments of d e present invention, the

elongation temperature selected for use in the PCR of the inventive methods is not more

tiian about 70°C. More preferably, the elongation temperature selected is between about 60°C and about 69°C, and even more preferably between about 65°C and about 69°C. Most

preferably, the elongation temperature employed in the PCR of the inventive methods is

about 68°C.

According to additional preferred embodiments of the present invention, die

denaturation temperature selected for use in d e PCR of the inventive methods is not more

than about 95°C. More preferably, the denaturation temperature selected is between about

90°C and about 95°C, and even more preferably between about 93°C and about 95°C. Most

preferably, the denaturation temperature employed in the PCR of the inventive methods is

about 94°C.

According to other preferred embodiments of the present invention, when the

tiiermal cycling includes an annealing temperature, the annealing temperature selected is

about 5-10°C below the melting temperature of the primers being employed. Preferably, die

annealing temperature selected is not more than about 65°C. More preferably, the annealing

temperature selected is between about 57°C and about 63°C, and even more preferably

between about 58°C and about 62°C. Most preferably, the annealing temperature employed

in the PCR of the inventive methods is about 60°C.

According to additional preferred embodiments of the present invention, during each

tiiermal cycle, d e amplification mixture is maintained at the elongation temperature for a

period of not less than about 1 minute. More preferably, during each thermal cycle, the

amplification mixture is maintained at the elongation temperature for a period of not less than about 2 minutes, and even more preferably for a period of not less than about 3

minutes.

According to particularly preferred embodiments of die present invention, the amplification mixture is maintained at the elongation temperature for a period of not less

d an about 2 minutes during die first cycle of the thermal cycling, and then the period during

which said amplification mixture is maintained at the elongation temperature is increased by

a period of about 5 seconds for each successive thermal cycle. Thus, for example, according to such embodiments of the present invention, if the amplification mixture was maintained

at die elongation temperature for a period of 2 minutes during the first cycle of the thermal

cycling, it would be maintained at the elongation temperature for a period of 2 minutes, 5

seconds for the second cycle, 2 minutes, 10 seconds for the third cycle, 2 minutes, 15

seconds for the fourth cycle, and so on until the thermal cycling is completed.

thermal cycle, the amplification mixture is maintained at the denaturation temperature for a

period of not more than about 1 minute. More preferably, during each tiiermal cycle, the

amplification mixture is maintained at the denaturation temperature for a period of not more

tiian about 45 seconds, and even more preferably for a period of not more than about 30

seconds, and still even more preferably for a period of not more than about 20 seconds.

Most preferably, during each thermal cycle, the amplification mixture is maintained at the

denaturation temperature for a period of not more than about 15 seconds, such as a period

of about 10 seconds. According to still other preferred embodiments of the present invention, when the thermal cycling includes an annealing temperature, the amplification mixture is maintained at

the annealing temperature for a period of not less than about 30 seconds. More preferably, according to such embodiments, during each thermal cycle, the amplification mixture is

maintained at the annealing temperature for a period between 30 seconds and 2 minutes, and

even more preferably for a period of not less than about 45 seconds. Most preferably,

during each thermal cycle, the amplification mixture is maintained at the annealing

temperature for a period of about 1 minute.

The number of thermal cycles employed in the PCR of the inventive metiiods may be

determined empirically by one skilled in the art depending, for example, on the suspected

concentration of the target sequence of interest in the biological material being tested.

According to preferred embodiments of die present invention, the amplification mixture is

subjected to at least about 30 cycles of tiiermal cycling, and even more preferably at least

about 40 cycles. Most preferably, the amplification mixture is subjected to at least about 50

cycles of d ermal cycling.

The polymerase employed in the PCR of die inventive metiiods may be any of die

suitable polymerases known to tiiose skilled in the art. Preferably, the polymerase employed

is a ti ermostable polymerase, i.e. a polymerase that is not adversely affected by the higher

temperatures involved in thermal cycling. More preferably, the polymerase may be a Taq

polymerase, or a suitable derivative thereof and/or a proof-reading polymerase.

According to particularly preferred embodiments of the present invention, at least

two polymerases are employed in the PCR of the inventive methods. Preferably, at least one of the polymerases is a Taq polymerase or a suitable derivative thereof, such as TaqMan DNA polymerase (available from Applied BioSystems), and the other polymerase is a proof¬

reading polymerase, such as ProofStart DNA polymerase (available from Qiagen).

According to certain preferred embodiments of the present invention, the amplification mixture further contains at least one thermostable inorganic pyrophosphatase.

Suitable amounts of thermostable inorganic pyrophosphatase may be determined empirically

by one skilled in art. Generally, when present, the ratio of thermostable inorganic

pyrophosphatase to Taq polymerase is at least about 1:20, more preferably at least about 1:10 and even more preferably at least about 1:5.

The remaining parameters employed in the PCR of the inventive methods, such as

d e primer concentration (generally about 100-500 nM and preferably about 200 nM)),

magnesium concentration (generally 1.5-6 mM and preferably about 1.5 mM of magnesium

sulfate and/ or magnesium chloride), deoxyribonucleotide triphosphates (dNTP)

concentration (generally about 0.2-0.4 mM each and preferably about 0.2 mM each), probe

concentration (if present, generally about 50-800 nM, and preferably about 100 nM), may

each be determined empirically by one skilled in the art using any of the known methods and

techniques.

According to certain particularly preferred embodiments of the present invention, the

deoxyribonucleotide triphosphates (dNTP) that are employed in d e PCR of d e inventive

methods are selected from the group consisting of C, T, G and A. Preferably, substantially

no dUTP is present in the amplification mixture of the inventive methods. According to still further preferred embodiments, substantially no uracil N-glycosylase is present in the amplification mixture of the inventive metiiods.

According to certain particularly preferred embodiments of the present invention, the amplification mixture futher comprises at least one buffer solution. Suitable buffer solutions

are known and available to those skilled in the art. Particularly preferred buffer solutions

include pH modifying buffers, such as buffers containing Tris-HCL and buffers which

maintain salt concentration, particular magnesium concentration, such as buffers containing

KC1 and/or (NH₄)₂S0₄.

After amplification using PCR, the first and second target nucleic acid sequences are

detected and quantified. This detecting and quantifying may be conducted using any of the

methods and techniques known to tiiose skilled in the art. For example, detecting and

quantifying of the first and second nucleic acid sequences may be conducted by adding a

suitable detecting agent, such as an intercalating dye, direcdy to the amplification mixture or

by adding a suitable nucleic acid probe to the mixture, preferably either a suitable nucleic

acid probe in combination with a detecting agent or a suitable nucleic acid probe having a

detectable label covalendy or ionically attached thereto or complexed therewith.

Preferably, the target nucleic acid sequence is detected by adding at least one nucleic

acid probe to d e biological material being tested. Any nucleic acid probe employed in the

inventive methods should contain sufficient nucleic acid residues to hybridizes selectively

under stringent conditions to a specific desired nucleic acid sequence, i.e. suitable probes will

generally contain at least 16 nucleic acid residues, and preferably hybridizes selectively under

stringent conditions to a specific nucleic acid sequence of the target nucleic acid sequence that is not the same as the nucleic acid sequence of any of the primers. Suitable nucleic acid probes include, but are not limited to, 5' nuclease probes, hairpin probes, adjacent probes,

sunrise probes and scorpion probes.

EXAMPLES

The following examples are illustrative, but not limiting, of d e present invention. Other suitable modifications and adaptations are of the variety normally encountered by

tiiose skilled in die art and are fully witiiin the spirit and scope of the present invention.

Example 1

Purpose: To demonstrate linear ampKfication of B19 DNA.

Materials: 1. B19 virus, titer 7.6 x 10¹¹ iu/ml from Bayer;

2. SNAP whole blood DNA isolation kit;

3. Forward Primer: Prism 5 (Figure 1) (SEQ ID NO.: 18);

4. Reverse Primer: Prism 6 (Figure 1) (SEQ ID NO.: 20);

5. Probe 3 (Figure 1) (SEQ ID NO.: 19) labeled with FAM at 5'

end and TAMRA at 3' end;

6. TaqMan Universal Master Mix, (ABI; cat. no. 4304437);

7. DNASE, RNASE free water;

8. ABI 96 weK plate and adhesive cores;

9. ANI 7000.

Procedure: 1. FoKowed SNAP protocol for extraction of 100 μl B 19

sample, eluted in 100 μl TE;

2. Diluted primers to 18 μM with TE;

3. Diluted probe to 5 μM with TE;

4. Prepared the foKowing master mix: TaqMan Master Mix: 25 μl;

Prism 5 (SEQ ID NO.: 18) 2.5 μl;

Prism 6 (SEQ ID NO.: 20) 2.5 μl;

Taqman Probe 2.5 μl;

Water: 12.54 μl;

5. Added 45 μl of master mix per weU;

6. SeriaUy diluted B19 DNA, adding water to the NTC weU;

7. Sealed and centrifuged the plate at 2300 rpm for about 30

seconds;

8. Ran PCR program for 50 cycles.

Results: A standard dilution curve was observed for B19 infected plasma,

vaKdating primer pair Prism 5 and Prism 6 (SEQ ID NOS.: 18 and 20) with Probe 3 (SEQ

ID NO.: 19).

Example 2

Purpose: To examine irradiated and unicradiated samples containing PPV using a

549 bp ampKcon.

Materials: 1. PPV (irradiated at 0 kGy, 50 kGy, 65 kGy, 75 kGy or 85

kGy);

2. SNAP Protein Degrader;

3. CeU Lysis Buffer; 4. Tris-HCl;

5. Primers: Prism 11 and Prism 12 (Figure 3) (SEQ ID NOS.: 40 and 42, respectively); and

6. Probe 6 (Figure 3) (SEQ ID NO.: 41).

Procedure: 1. To 100 μl viral sample, added 50 μl tris-HCl buffer, 60 μl

protein degrader, and 200 μl ceU lysis buffer;

2. Mixed and incubated for 25 minutes (5 minutes at 70°C);

3. Diluted samples to 1/50, 1/500, 1/5000, 1/25000, 1/50000,

1/250000 and 1/500000;

4. Ran PCR for 55 cycles.

Results: Results showed that unirradiated material had regular dilution series

curves, irradiated material (50 kGy) behaved differently, dilute material did not ampKfy

showing a reduction in the number of copies of the target sequence.

Example 3

Purpose: To determine effects of gamma irradiation (0 kGy sample, 50 kGy

sample, mixture of 0+50kGy sample and 75 kGy sample) on samples containing PPV

analyzed by PCR.

Materials: 1. PPV (irradiated at 0 kGy, 50 kGy or 75 kGy);

2. Primers: Prism 11 & Prism 12, Probe 6 (Figure 3) (SEQ ID

NOS.: 40, 42 and 41, respectively); 3. Primers: Prism 1 & Prism 2, Probe 1 (Figure 3) (SEQ ID NOS.: 43, 45, and 44, respectively).

Procedure: 1. Diluted samples containing PPV to 1/100, 1/1000, 1-2000,

1/10000, 1/20000, 1/40000 and 1/400000 (0 kGy, 50 kGy, 0+50 kGy and 75 kGy);

2. Ran PCR program for 55 cycles.

Results: Irradiaition to 50 kGy of PPV material reduced ampKfication of 549 bp

ampKcon.

Example 4

Purpose: To examine the relative effectiveness of Qiagen and Taqman reagents

on samples containing PPV.

Materials: 1. PPV DNA (phenol extracted);

2. Taq PCR Core Kit;

3. ProofStart DNA polymerase;

4. Taqman Universal PCT Master Mix;

5. Prism 1, 2, 11 and 17 (Figure 3) (SEQ ID NOS.: 43, 45, 40,

and 47 respectively);

6. Probes 1 and 6 (Figure 3) (SEQ ID NOS.: 44 and 41,

respectively);

7. Agarose;

8. TAE;

9. EtBr. Procedure: 1. Prepared the foUowing four master mixes:

a. Qiagen: 1 2

lOx buffer: 30 μl 25 μl

dNTP's: 9 μl 7.5 μl

pA: 8.34 μl 6.95 μl

pB: 8.34 μl 6.95 μl

taq: 6 μl 5μl

H₂0: 187.32 μl 156.1 μl

probe: 15 μl 12.5 μl

b. Taqman: 3 4

Master Mix: 150 μl 125 μl

pA: 15 μl 12.5 μl

pB: 15 μl 12.5 μl

probe: 15 μl 12.5 μl

H₂0: 69 μl 57.5 μl

2. Pipetted 44 μl of master mix 1 into row D, weKs 1 and 2;

row E, weUs 1 and 2; and row H, weU 1, of a weK plate;

3. Pipetted 44 μl of master mix 2 into row D, wells 3 and 4;

and row E, wells 3 and 4, of a weU plate;

4. Pipetted 44 μl of master mix 3, into row F, weKs 1 and 2;

row G, weKs 1 and 2; and row H, weK 3, of a weK plate; 5. Pipetted 44 μl of master mix 4 into row F, weKs 3 and 4; and

row G, weKs 3 and 4, of a weK plate;

6. Added 1 μl of ProofStart taq to row D, weKs 1-4 and row F,

wells 1-4 and added 1 μl water to remaining weKs;

7. Added 5 μl water to row H, wells 1 and 3 and added 5 μl

PPV DNA to remaining weKs;

8. Ran PCR for 40 cycles.

Results: Qiaqen Master with ProofStart taq produced functional large ampKcons in realtime PCR with PPV DNA more efficiently than the TaqMan master mix.

Example 5

Purpose: To examine the effects of proofstart in ampKfying large ampKcons and

to examine the effects of 50 kGy irradiation on PPV.

Materials: 1. PPV DNA (irradiated to 0 kGy and 50 kGy);

2. Taq PCR Core Kit;

3. Proofstart DNA polymerase;

4. Prism 11, 16 and 17 (Figure 3) (SEQ ID NOS.: 40, 46 and

47, respectively);

5. Agarose;

6. Ethidium Bromide;

7. TAE buffer.

Procedure: 1. Set up PCR master mix as foKows: lOx buffer: 50 μl

dNTP's: 15 μl

pA: 13.9 μl (primer 11)

taq: 10 μl

water: 347.2 μl

2. Placed aKquots into PCR tubes;

3. Added either primer 16 or 17 to PCR tubes;

4. Added PPV DNA (dKuted to 1:100) to each PCR tube:

5. Added 10 μl proofstart to half of the samples (2 at 0 kGy

and 2 at 50 kGy);

6. Performed PCR (about 55 cycles)

7. Poured a 1% gel and ran at 100 V for 20 minutes.

Results: Addition of a proofreading polymerase resulted in improved

ampKcation of longer ampKcons. Delay in ampKfication of target sequence in irradiated

samples is proportional to damage done to viral genetic material.

Example 6

Purpose: To examine the effect of TSP concentration on ampKfication of large

target ampKcons in gamma irradiated and unirradiated PPV.

Materials: 1. TSP (cat. no. M02965);

2. Qiagen Core kit; 3. ProofStart DNA polymerase;

4. PPV (irradiated to 0 kGy or 50 kGy).

Procedure: 1. Prepared a master mix (standard PCR set-up) for each

(TSP Taq 1:20, 1:10, 1:5);

Added 43.61 μl of each master mix (TSP titration) to

PCR tubes;

3. Added 1.39 μl of primers 16, 17 or 19 (Figure 3) (SEQ

ID NOS.: 46, 47, and 49, respectively) to appropriate PCT tubes;

4. Added 5 μl water to the negative control, which

contained primer pair 11, 16 (Figure 3) (SEQ ID NOS.: 40 and 46, respectively);

5. Diluted PPV 1:100;

6. Added PPV to PCR tubes;

7. Performed PCR;

8. Poured a 1% gel and ran at 100 V for 20 minutes.

Results: Under these conditions, addition of TSP resulted in increased

ampKfication of target ampKcons in both irradiated and unirradiated samples, but irradiation

of PPV resulted in decreased ampKfication of target ampKcons.

Example 7

Purpose: To examine the effects of gamma irradiation on ampKfication of PPV

target ampKcons of various sizes.

Materials: 1. PPV DNA (irradiated to 0 kGy or 50 kGy); 2. Taq PCR Core Kit;

3. ProofStart DNA Polymerase;

4. Prism 11, 16, 17, 18 and 19 (Figure 3) (SEQ ID NOS.: 40, 6, 47, 48, and 49, respectively);

5. Agarose;

6. TAE;

7. Ethidium Bromide.

Procedure: 1. Prepared PCR Master Mix as foKows:

1 Ox Buffer 5 μl

dNTPs 1.5 μl

pA 1.39 μl

pB 1.39 μl

taq 1 μl

water 33.72 μl

PPV 5 μl.

2. AlKquoted samples into PCR tubes;

3. Ran PCR;

4. Poured a 1% agarose gel and ran at 120 V for about 1.5

hours.

Results: Irradiation to 50 kGy resulted in decreased amplification of larger

target ampKcons. Example 8

Purpose: To examine PCR sensitivity and determine log reduction of PPV in samples irradiated to 50 kGy and having a starting concentration of 2.5x10⁷ gEq.

Materials: 1. Standard PCR reagents (Qiacen Core Kit, TSP, Proofstart,

etc.);

2. Primers 11 and 17 (Figure 3) (SEQ ID NOS.: 40 and 47, respectively);

3. PPV extract.

Procedure: 1. Prepared master mix with primers 11 and 17;

2. Performed a 10 fold dilution series from 10⁷ to 10° of PPV

extract;

3. Pipetted 45 μl of master mix into PCR tubes;

4. Pipetted 5 μl of each PPV dKution into appropriated PCR

tubes;

5. Added 5 μl water to control;

6. Ran PCR;

7. Ran samples in 1% agarose at 100V for about 47 minutes.

Results: Irradiation of sample to 50 kGy resulted in decreased ampKfication of

target ampKcon across aK concentration ranges. Example 9

Purpose: To examine PCR sensitivity and determine log reduction of PPV

irradiated to 50 kGy and having a starting concentration of 2.5xl0⁷ gEq.

Materials: 1. TSP;

2. Standard PCR kit (Qiacen with ProofStart Polymerase);

3. Primers 11 and 19 (Figure 3) (SEQ ID NOS.: 40 and 49,

respectively);

4. PPV Extract (Irradiated to 0 kGy and 50 kGy). Procedure: 1. Prepared master mix with primers 11 and 19 (SEQ ID

NOS.: 40 and 49, respectively);

2. Performed a 10 fold dilution series from 10⁷ to 10° of PPV

extract;

3. Pipetted 45 μl of master mix into PCR tubes;

4. Pipetted 5 μl of each PPV dilution into appropriate PCR

tubes;

5. Added 5 μl water to control;

6. Ran PCR as foKows: 95°C for 2 minutes (1 cycles)

94°C for 10 seconds (40 cycles)

60°C for 1 minute (40 cycles)

68°C for 2 minutes (40 cycles);

7. Cooled to 4°C; 8. Ran samples on 1% agarose gel in lx TAE and 5 μl/100 ml

ethidium bromide at 100 V for 52 minutes (5 μl on gel).

Results: Irradiation to 50 kGy resulted in decreased amplification of target ampKcon across aK concentration ranges. For unirradiated samples, relative band strength of

observed target ampKcon decreased with decreasing concentration.

Example 10

Purpose: Primer vaKdation for B19 using probe 7 (SEQ ID NO.: 12) and

various primers.

Materials: 1. Bl 9 IGIV Paste (irradiated to 0 kGY or 50 kGy);

2. EXB;

3. Proteinase;

4. yeast tRNA

5. phenol chloroform isoamyl alcohol;

6. 3M NaAc;

7. isopropanol;

8. 70% EtOH;

9. TE buffer;

10. Prisms 5, 6, 20, 21, 22, 23, 24, 25, 26 (Figure 1) (SEQ ID

NOS.: 18, 20, 11, 13, 14, 15, 16, 17, and 21, respectively);

11. Qiagen reagents;

12. AmpKgold Taq; 13. ProofStart Polymerase;

14. Agarose;

15. TAE;

16. Ed idium Bromide.

Procedure: 1. Prepared a Master Mix as foKows:

Buffer 5 μl

DNTP 1.5 μl

Taq 1 μl

DNA 5 μl

water 34.72 μl

2. Pipetted Master Mix into PCR tubes;

3. Added the foKowing primer pairs to appropriate PCR tubes:

20&21 (SEQ ID NOS.: 11 and 13, respectively); 20&22 (SEQ ID NOS.: 11 and 14,

respectively); 20&23 (SEQ ID NOS.: 11 and 15, respectively); 20&24 (SEQ ID NOS.: 11

and 16, respectively); 20&25 (SEQ ID NOS.: 11 and 17, respectively); 20&6 (SEQ ID NOS.

11 and 20, respectively); 20&26 (SEQ ID NOS.: 11 and 21, respectively); 5&6 (SEQ ID

NOS.: 18 and 20, respectively);

4. Ran PCR;

5. ran 1% gel for about 1 hour.

Results: AK tested primers yielded desired target ampKcons. Example 11

Purpose: Use of PCR multiplexing with target ampKcons of about 112 bp and

about 2.4 kbp for B19 virus in samples irradiated to 0 kGy or 50 kGy.

Materials: 1. TSP thermostable inorganic pyrophosphatase

2. Standard PCR reagents;

3. B19 viral extract (irradiated to 0 kGy and 50 kGy);

4. Prisms 5, 6, 20 and 25 (Figure 1) (SEQ ID NOS.: 18, 20, 11

and 17, respectively);

5. Taq;

10 6. ProofStart Polymerase.

Procedure: 1. Prepared standard PCR set-up with 3x master mixes, for

each primer set (primer sets: 5&6 (SEQ ID NOS.: 18 and 20, respectively); 20&25 (SEQ ID

NOS.: 11 and 17, respectively); 5&6 (SEQ ID NOS.: 18 and 20, respectively); and 20&25

(SEQ ID NOS.: 11 and 17, respectively));

15 2. Prepared appropriate PCR tubes containing the foKowing

primer pairs: (5, 6) 0 kGy; (5, 6) 50 kGy; (20, 25) 0 kGy; (20, 25) 50 kGy; (5, 6) & (20, 25), 0

kGy; and (5, 6) and (20, 25), 50 kGy;

3. Added 5 μl B19 to PCR tubes containing 45 μl of

appropriate master mix;

>0 4. Added 5 μl water to control;

5. Ran PCR. 6. Ran samples on 1% aragose gel at 100 V for about 17

minutes.

Results: PCR multiplexing is effective for mixtures containing large target ampKcons and small target ampKcons. Irradiation to 50 kGy resulted in decreased

ampKfication of the large target ampKcon relative to the small target ampKcon.

Example 12

Purpose: Irradiated and unirradiated samples containing B19 viral material were

examined using real time PCR.

Materials: 1. B19 viral material (irradiated to 0 kGy and 50 kGy);

2. Prism pairs (20, 21) (SEQ ID NOS.: 11 and 13, respectively)

and (20, 26) (SEQ ID NOS.: 11 and 21) (Figure 1);

3. Qiagen PCR reagents;

4. Qiagen ProofStart;

5. Agarose;

7. sample loading buffer (SLB).

Procedure: 1. Prepared standard samples containing primer pairs with 10¹¹

to 10¹ dKution series;

2. Ran PCR (40 cycles);

3. Ran gel on 1% agarose (8 μl PCR product, 1 μl SLB) at 100

V for about 20 minutes. Results: Irradiation to 50 kGy resulted in decreased amplification of large target ampKcon. Unirradiated samples exhibited a regular dKution pattern.

Example 13

Purpose: To investigate the effe ct of gamma irradiation on samples containing

HBV and irradiated to 50 kGy.

Materials: 1. HBV (irradiated to 0 kGy and 50 kGy);

2. Taq PCR Core Kit;

3. ProofStart DNA polymerase;

4. Prisms 34, 9, 10, 15, 29, 30, 31, 36 and 37 (Figure 2) SEQ ID

NOS.: 31, 22, 24, 25, 27, 32, 34, 28, and 29, respectively);

5. Agarose;

6. TAE Buffer;

7. ethidium bromide.

Procedure: 1. Prepared PCR master mix as foKows:

lOx PCR buffer 5 μl

dNTPs 1.39 μl

primers 1.39 μl

taq l μl

ProofStart 1 μl

water 33.22 μl TSP 0.5 μl

2. Aliquoted 43.61 μl of master mix into PCR tubes.

Appropriate tubes contained the foKowing primer pairs: (3, 4); (9, 10); (9, 15); (9, 29); (9, 30);

(9, 31); (36, 37); and (9, 31), for both irradiated and unirradiated samples;

3. Added 5 μl HBV per tube (irradiated or unirradiated);

4. Ran PCR as foKows:

50°C for 2 minutes (one cycle) 95°C for 2 minutes (one cycle)

94°C for 10 seconds (40 cycles)

60°C for 1 minute (40 cycles)

68°C for 2 minutes, five seconds (40 cycles);

5. Ran 1% agarose gel (9 μl sample + 1 μl sample buffer) at

lOOv for about 20 minutes.

Results: Irradiated samples showed no ampKfication of large target ampKcons,

indicating degradation of HBV genetic material by irradiation to 50 kGy.

Example 14

Purpose: To investigate the effect of gamma irradiation on samples containing

HBV DNA and irradiated to 50 kGy.

Materials: 1. HBV DNA material (irradiated to 0 kGy and 50 kGy);

2. Taq PCR Core Kit (Qiagen, cat. no. 201223);

3. ProofStart Taq Polymerase (Qiagen, cat. no. 20); 4. Prisms 10, 13, 30, 36 and 37 (Figure 2) (SEQ ID NOS.: 24,

6, 32, 28, and 29, respectively);

5. Agarose;

6. TAE Buffer;

7. Ethidium Bromide.

Procedure: 1. Prepared the foKowing master mix:

lOx buffer 60 μl

dNTP 18 μl

primer 36 (SEQ ID NO.: 28) 16.68 μl

Taq 12μl

ProofStart 12 μl

water 440.64 μl;

2. Pipetted 46.61 μl of master mix into PCR tubes;

3. Added 1.39 μl of reverse primer (10, 13, 30 or 37) (SEQ ID

NOS.: 24, 26, 32, and 29, respectively) and 2 μl HBV DNA (0 kGy and 50 kGy) to

appropriate tubes;

4. Ran PCR for 50 cycles;

5. Poured a 1% agarose gel (8 μl PCR product + 1 μl sample

buffer) at 100 V for about 20 minutes.

Results: Irradiated samples showed no ampKfication of large target ampKcons,

indicating degradation of HBV DNA by irradiation to 50 kGy. Example 15

Purpose: HBV ampKfication of nested primer set (about 80 bp, 400 bp and 697 bp) in samples containing ascorbate, including digestion of 0 kGy and 50 kGy samples witii

5 exonuclease I prior to PCR ampKcation.

Materials: 1. HBV DNA (irradiated to 0 kGy and 50 kGy, with and without ascorbate);

2. Primer sets: (9, 10) (SEQ ID NOS.: 22 and 24, respectively);

(9, 15) (SEQ ID NOS.: 22 and 25, respectively); and (9, 13) (SEQ ID NOS.: 22 and 26,

0 respectively) (Figure 2);

3. Exonuclease I;

4. Standard PCR reagents.

Procedure: 1. DKuted HBV samples to 1/500, 1/2000 and 1/10000;

2. Digested 1 μl raw HBV extract in 0.25 μl Exonuclease I, 10

5 μl lOx Exonuclease I buffer and 88.75 μl water at 37°C for 30 minutes, inactivated at 80°C

for 20 minutes;

3. DKutes digested HBV to 1/2000 and 1/10000;

4. Ran 55 cycles PCR.

Results: Irradiated showed no ampKfication of large target ampKcon (697 bp),

:0 indicating degradation of HBV DNA by irradiation to 50 kGy. Example 16

Purpose: To investigate the amount of bacterial and fungal DNA present in

pulverized tendon samples.

Materials: 1. E. CoK samples (tendon) — 0 or 50 kGy + stabiKzer

(6.65x10¹⁰ CFU/μl);

2. C. Albicans samples (tendon) - 0 or 50 kGy + stabiKzer

(3.55x10⁹ CFU/μl);

3. Staph. Aureus samples;

4. Control tendon;

5. Dneasey tissue kit (Qiagen, cat. no. 69504);

6. Taq PCR Core Kit (Qiagen, cat. no. 201223);

7. ProofStart Taq Polymerase (Qiagen, cat. no. 202205);

8. Primers: Ribo 7 and 8, and Ribo 10, 11, 12, 13, 14 (Figures

6A and 6B) SEQ D NOS.: 69, 70, 71, 72, and 73, respectively); and Fungi 1, 2, 3, 4, 5, 6, 7, 8

(Figures 7A and 7B) (SEQ ID NOS.: 75, 77, 78, 79, 80, 81, 82, and 83 respectively);

9. Probes: FAM-RIBO

Fungi Probe (Figure 7A) (SEQ ID NO.: 76) labeled with

FAM at 5' end and TAMRA at 3' end;

10. Microcon YM Centrifugal Filter Unit;

Procedure: 1. Using 0.05 tendon samples for E. coK and C. albicans,

followed the Qiagen extraction profile;

2. Prepared the foKowing master mixes: Mix 1 Mix 2

lOx buffer 150 μl 85 μl

dNTPs 45 μl 25.5 μl

Ribo 7 41.7 μl

Fungi 1 (SEQ ID NO. 75) — 23.65 μl

Taq 30 μl 17 μl

ProofStart 30 μl 17 μl

Water 936.6 μl 530.74 μl

FAM-RIBO 75 μl

Fungi Probe — 42.5 μl

3. FKtered master mixes using Microcon filter units for 30

minutes at 1 OOx g;

4. Pipetted 43.6 μl of Mix 1 into: rows A-D, columns 1-6; rows

A-C, column 9; and row E, column 12;

5. Pipetted 43.6 μl of Mix 2 into: rows E-F, columns 1-7 and

row H, column 12;

6. Pipetted 1.39 μl of reverse primer into appropriate weK;

7. Pipetted 5 μl DNA into appropriate weKs;

8. Ran PCR. Results: Irradiation with 50 kGy resulted in decreased ampKfication of large target ampKcons, indicating degradation of the pathogen genetic material caused by

irradiation.

Example 17

Purpose: To show functionaKty of E. coK primers for RT-PCR using large target

ampKcons.

Materials: 1. E. coK prepared from overnight culture;

2. Dneasy Tissue Kit (Qiagen, cat. no. 96504);

3. Taq PCR Core Kit (Qiagen, cat. no. 201223)

4. ProofStart DNA polymerase (Qiagen, cat. no. 202205);

5. Microcon YM-100 Centrifugal FKter Unit (cat. no. 42413);

6. Primers: Ribo 1-6 (SEQ ID NOS.: 62, 64, 65, 66, 67, and

68, respectively) and Ribo 7-9;

7. Agarose;

8. TAE Buffer;

9. Ethidium Bromide.

Procedure: 1. Pipetted 1 ml of E. coK culture into each of 10 1.5 tubes;

2. Centrifuged aK 10 tubes for 5 minutes at maximum speed;

3. Discarded supernatant;

4. Placed 8 tubes in -80°C and used 2 tubes for extraction

foKowing the Qiagen protocol; 5. Prepared Master Mix as foKows:

1 Ox Buffer 5 μl

dNTPs 1.5 μl

pA 1.39 μl (Ribo 1 (SEQ ID NO.: 62))

5 or (Ribo 7)

pB 1.39 μl (Ribo 2, 3, 4, 5, or 6) (SEQ ID

NOS.: 64, 65, 66, 67, or 68, respectively) or (Ribo 8 or 9)

Taq 1 μl

ProofStart 1 μl

0 Water 33.22 μl

TSP 0.5 μl

6. Mixed Master Mix by inversion;

7. Pipetted Master mix into a Microcon Centrifugal Filter Unit

and centrifuged for 30 minutes at lOOx g;

5 8. Pipetted 43.61 μl of Master Mix into PCR tubes;

9. Added appropriate reverse primer and DNA or water to

create the foKowing primer pairs: (1, 2) + 5 μl DNA; (1, 2) + 1 μl; (1, 3) + 5 μl DNA; (1, 3)

+ 1 μl DNA; (1, 4) + 5 μl DNA; (1, 4) + 1 μl DNA; (1, 5) + 5 μl DNA; (1, 5) + 1 μl DNA;

(1, 6) + 5 μl DNA; (1, 6) + 1 μl DNA; (5, 8) + 5 μl DNA; (7, 8) + 1 μl DNA; (7, 9) + 5 μl

>0 DNA; (7, 9) + 1 μl DNA; and (1, 2) + 5 (1, 4) + 5 μl water;

10. Ran PCR;

11. Ran 1 % Agarose gel at 100 V for about 20 min. Results: AK E. coK primers showed ampKfication of target sequences, regardless of size.

Example 18

Purpose: To investigate the effects of 50 kGy irradiation on samples containing

E. coK.

Materials: 1. E. coK spiked tendon (irradiated to 0 kGy and 50 kGy)

2. Taq PCR Core Kit (Qiagen, cat. no. 201223);

3. ProofStart Taq Polymerase (Qiagen, cat. no. 202205);

4. Primers: Ribo 7 and 8, and Ribol3, 14 and 15 SEQ ID

NOS.: 72, 73, and 74, respectively);

5. Agarose;

6. TAE Buffer;

7. Ethidium Bromide;

8. Microcon Centrifugal FKter Unit.

Procedure: 1. Prepared Master Mix as foKows:

1 Ox Buffer 60 μl

dNTP 18 μl

pA (forward) 16.68 μl

Taq 12 μl ProofStart 12 μl

Water 452.64 μl;

2. Placed in Microcon and centrifuged for 30 minutes at lOOx g;

3. Pipetted 47-61 μl master mix into each or 9 PCR tubes;

4. Added 1.39 μl of reverse primer and 1 μl DNA into

appropriate tubes;

5. Ran PCR.

6. Ran 1% Agarose gel (8 μl sample + 1 μl sample buffer) at

100 V for about 20 minutes.

Results: Samples irradiated to 50 kGy showed progessive disappearance of

bands witii increasing ampKcon size, indicating degradation of d e E. coli genetic material

caused by irradiation.

Example 19

Purpose: To show functionaKty of Mt-DNA primers for RT-PCR using large

target ampKcons.

Materials: 1. Tendon DNA (irradiated to 0 kGy and 50 kGy);

2. ROX 6 (1/10 dKution) molecular probes;

3. Primers: MITO 1, 2, 3, 4, and 5 (Figure 8) (SEQ ID NOS.

90, 92, 95, 96, and 97, respectively);

4. MITO Probe 1 (Figure 8) (SEQ ID NO.: 91); 5. Human DNA;

6. Qiagen PCR Reagants;

7. Qiagen ProofStart. Procedure: 1. Prepared the foKowing mixtures:

Buffer 1.5 μl

dNTPs 1.5 μl

MITO 1 2.5 μl

reverse primer 2.5 μl (MITO 2, 3, 4 or 5)

MITO Probe 2.5 μl

Taq 1 μl

PS 1 μl

1/10 ROX 1 μl

water 28 μl

DNA 5 μl

2. Ran 40 PCR;

3. Ran 1% agarose gel (8 μl product + 1 μl sample loading

buffer) at 100 V for about one hour.

Results: Mt-DNA primers were functional, regardless of ampKcon size.

Example 20

Purpose: Real-time PCR ampKfication of human DNA (large ampKcons). Materials: 1. 10 ng of human control DNA; Calbiochem, Human

Genomic DNA, Cat #HCD01, Lot # D10498;

2. Taq PCR Core Kit;

3. ProofStart DNA Polymerase;

4. Primers and Probes;

5. Agarose;

6. TAE;

7. Ethidium Bromide.

Procedure: 1. Prepared PCR Master Mix as foKows:

1 Ox Buffer 5 μl

dNTPs 1.5 μl

Mito 1 (SEQ ID NO. 90) 2.5 μl

Reverse Primer (Mito 5 or 7)

(SEQ ID NO. 97 or 99, respectively) 2.5 μl

MitoProbe 1 (SEQ ID NO. 91) 2.5 μl

taq 1 μl

Proof Start 1 μl

water 31 μl

water or DNA 3 μl

2. Ran PCR (50 cycles); 3. Ran 8 μl PCR Products on 1% agarose gel and ran at 100 V

for about 20 minutes.

Results: Target sequences greater then 8,000 nucleic acid residues can be

successfuKy ampKfied with Real-time PCR.

Example 21

Purpose: Real-time PCR on fibular bone rings to detect bacterial contamination

in unirradiated bone samples.

Materials: 1. Bacterial extracts from bones;

2. Taq PCR Core Kit (Qiagen, Cat#201223);

3. ProofStart DNA Polymerase (Qiagen, Cat#202205);

4. Primers: Ribo 7 and 10 (SEQ ID NOS. 100 and 69,

respectively);

5. Probe: FAM-RIBO (SEQ ID NO. 101);

6. OpticaKy clear plates and seals;

Procedure: 1. Prepared PCR setup as foKows:

Per run x23

1 Ox Buffer 5 μl 115 μl

dNTPs 1.5 μl 34.5 μl

pA 3.5 μl 80.5 μl pB 3.5 μl 80.5 μl

Probe 2.5 μl 57.5 μl

taq 0.25 μl 5.75 μl

Proof Start 1 μl 23 μl

water 30.75 μl 707.25 μl

2. AKquot 48 μl into A4-7, B4-7, C4-7, D4-7, and Hll-12;

3. Pipet 2 μl of appropriate DNA into each weK

4. Seal plate and run "long" program on the thermocycler (40

cycles).

Results: Of 103 bone samples, 40% were found to be contaminated with

bacteria.

Having now fuKy described this invention, it wiK be understood to those of ordinary

skiK in the art that the methods of the present invention can be carried out with a wide and

equivalent range of conditions, formulations and other parameters without departing from

the scope of the invention or any embodiments thereof.

AK patents and pubKcations cited herein are hereby fuKy incorporated by reference in

tiieir entirety. The citation of any pubKcation is for its disclosure prior to d e filing date and

should not be construed as an admission that such pubKcation is prior art or that the present

invention is not entitled to antedate such pubKcation by virtue of prior invention.

The foregoing embodiments and advantages are merely exemplary and are not to be

construed as Kmiting the present invention. The present teaching can be readily appKed to

other types of apparatuses. The description of the present invention is intended to be Klustrative, and not to limit the scope of the claims. Many alternatives, modifications, and

variations wKl be apparent to those sk led in the art. In the cla ns, means-plus-function

clauses are intended to cover the structures described herein as performing the recited function and not only structural equivalents but also equivalent structures.

Claims

WHAT IS CLAIMED IS:

1. A method for analyzing a target nucleic acid sequence in a biological material, said method comprising:

5 (i) adding to said biological material an effective amount of at least

two nucleic acid primers,

wherein said nucleic acid primers hybridize under stringent

conditions to predetermined nucleic acid sequences of said target nucleic acid sequence that

are separated by at least about 750 nucleic acid residues,

[0 (K) ampKfying said target nucleic acid sequence by polymerase chain

reaction, said polymerase chain reaction comprising adding a polymerase to said biological

material and primers to form an ampKfication mixture and thermaKy cycKng said

ampKfication mixture between at least one denaturation temperature and at least one

elongation temperature,

5 wherein said elongation temperature is not more than about

70°C and said denaturation temperature is not more than about 95°C, and further wherein

during each thermal cycle said ampKfication mixture is maintained at said denaturation

temperature for a period of not more than about 30 seconds and at said elongation

temperature for a period of not less than about 1 minute; and

0 (Ki) detecting and quantifying said target nucleic acid sequence.

2. The method according to claim 1, wherein said predetermined nucleic acid sequences of said target nucleic acid sequence are separated by at least about 1000 nucleic acid residues of said target nucleic acid sequence

3. The method according to claim 1, wherein said predetermined nucleic acid sequences of said target nucleic acid sequence are separated by at least about 2000 nucleic acid residues of said target nucleic acid sequence I

4. The method according to claim 1, wherein said predetermined nucleic acid sequences of said target nucleic acid sequence are separated by at least about 3000 nucleic acid residues of said target nucleic acid sequence

5. The method according to claim 1, wherein said predetermined nucleic acid sequences of said target nucleic acid sequence are separated by at least about 4000 nucleic acid residues of said target nucleic acid sequence.

6. The method according to claim 1, wherein said predetermined nucleic acid sequences of said target nucleic acid sequence are separated by at least about 5000 nucleic acid residues of said target nucleic acid sequence.

7. The method according to claim 1, wherein said predetermined nucleic acid sequences of said target nucleic acid sequence are separated by only about 500 nucleic acid residues of said target nucleic acid sequence.

8. The method according to claim 1, wherein said step (i) further comprises adding at least one nucleic acid probe to said biological material.

9. The method according to claim 8, wherein said nucleic acid probe is selected from d e group consisting of 5' nuclease probes, hairpin probes, adjacent probes, sunrise probes and scorpion probes.

10. The method according to claim 1, wherein said elongation temperature is between about 60°C and about 69°C.

11. The method according to claim 1, wherein said elongation temperature is between about 65°C and about 69°C.

12. The method according to claim 1, wherein said denaturation temperature is between about 90°C and about 95°C.

13. The method according to claim 1, wherein said denaturation temperature is between about 93°C and about 95°C.

14. The method according to claim 1, wherein during each thermal cycle said ampKfication mixture is maintained at said denaturation temperature for a period of not more tiian about 20 seconds.

15. The method according to claim 1, whereki during each thermal cycle said ampKfication mixture is maintained at said denaturation temperature for a period of not more tiian about 10 seconds.

16. The method according to claim 1, wherein during each thermal cycle said ampKfication mixture is maintained at said elongation temperature for a period of not less d an about 2 minutes.

17. The method according to claim 1, wherein during each thermal cycle said ampKfication mixture is maintained at said elongation temperature for a period of not less than about 3 minutes.

18. The method according to claim 1, wherein the period during which said ampKfication mixture is maintained at said elongation temperature during each thermal cycle is increased by a period of about 5 seconds for each successive thermal cycle.

19. The method according to claim 1, wherein said ampKfication mixture is tiiermaKy cycled for at least 30 cycles.

20. The method according to claim 1, wherein said ampKfication mixture is tiiermaKy cycled for at least 40 cycles.

21. The method according to claim 1, wherein said ampKfication mixture is tiiermaKy cycled for at least 50 cycles.

22. The method according to claim 1, wherein said biological material has been subjected to an environment or process that may have altered said target nucleic acid sequence.

23. The method according to claim 1, wherein said polymerase is a Taq polymerase.

24. The method according to claim 1, wherein said polymerase is a proof-reading Taq polymerase.

25. The method according to claim 1, wherein said ampKfication mixture further comprises at least one thermostable inorganic pyrophosphatase.

26. The method according to claim 25, wherein the ratio of Taq polymerase to thermostable inorganic pyrophosphatase is about 5:1.